Members of the NF-?B/Rel family of transcription factors are well-established as critical regulators of pro- inflammatory genes in cells of the innate immune system, and they play important roles in several other biological processes. The phenotypes of mice lacking individual NF-?B family members suggest that each protein targets unique sets of genes. However, although much has been learned about the signal transduction mechanisms that activate NF-?B dimers in response to various stimuli, the fundamental reasons different NF-?B dimers are capable of regulating different genes remain largely unexplored. Because some genes that require a specific NF-?B family member for expression play critical roles in dictating the type of immune response elicited by a stimulus, an understanding of the mechanistic basis of family member selectivity may lead to novel strategies for modulating immune responses with an unusually high degree of specificity, to more effectively combat infectious diseases and chronic inflammatory diseases. We became interested in the mechanisms of NF-?B family member selectivity when we found that the II12b gene, which encodes the p40 subunit of the pro-inflammatory cytokine IL-12, exhibits an unusually strong requirement for the c-Rel member of the NF-?B family. Because earlier studies had shown that the DNA- contacting residues of c-Rel are identical to those of another family member, p65, and that the DNA recognition sequences for c-Rel and p65 are similar, we asked why c-Rel, but not p65, is critical for Il12b activation. Our results provided compelling evidence that c-Rel is required because c-Rel homodimers can bind non-consensus NF-?B sequences with high affinity, whereas p65 homodimers bind with high affinity only to sequences that closely match the NF-?B consensus. Notably, 46 residues of c-Rel were found to be responsible for its unique DNA-binding properties. To further elucidate NF-?B selectivity mechanisms, we will first use a variety of interconnected strategies to advance our knowledge of gene activation by c-Rel. We will also prepare bacterial artificial chromosome (BAC) transgenic mice to examine more rigorously the hypothesis that the 46 residues of c-Rel are fully responsible for its unique functions. Finally, because of the success of our p65-c-Rel chimeric protein strategy for uncovering c-Rel selectively mechanisms, we will use this same strategy as a starting point toward understanding the mechanism of selective gene activation by p65. [unreadable] [unreadable] [unreadable]